Agent for use in the case of fructose intolerance

ABSTRACT

There is provided in accordance with embodiments of the invention a method of treating or reducing the effects in a subject of a condition selected from fructose intolerance and impaired fructose metabolism, the method comprising administering to a subject in need of such treatment or reduction an efficacious amount of a glucose isomerase, other than in combination with 5-D-fructose dehydrogenase. Other embodiments are also disclosed.

This application is a continuation-in-part of PCT/EP2008/001294, filedFeb. 20, 2008, which claims priority from DE 10 2007 008 664.6, filedFeb. 20, 2007. The contents of both of these applications areincorporated herein by reference.

This patent application discloses an agent for use in the case offructose intolerance, which contains a compound that effects theconversion of fructose to glucose. The term fructose intolerance is usedin the context of this patent application to mean not only the medicallydefined fructose intolerance and fructose metabolism disorder (seebelow), but any form of impairment of health or well-being that occursas a result of the intake of fructose or fructose containing foodstuffs,or due to the release of fructose in the digestive tract of humans oranimals from other substances, such as sucrose.

In the context of this patent application, the terms “food” and“foodstuff” are used as synonyms. They mean to also include feed in thesense of animal feed.

Fructose is a ketohexose and is an important energy providing ingredientof food. It is present in numerous foodstuffs, as a component of di- andoligosaccharides, and/or as free fructose. Food such as fruit and fruitjuices contains large amounts of fructose, but in particular alsosucrose, which is cleaved to fructose and glucose in the body. In thefollowing, the term ‘fructose containing’ is used to mean all substancesand foodstuffs that either contain fructose as such or in a form fromwhich fructose can be released in the digestive tract. The ‘fructosecontent’ of substances and foodstuffs refers to all the fructose in afructose containing food or substance in whatever form it is containedin such a food or substance (free and also e.g. as part of sucrose).

In contrast to glucose, fructose is assimilated into the mucosa cells ofthe small intestine by eased carrier-mediated diffusion. The enzymaticdegradation starts in the liver by the action of the adenosinetriphosphate (ATP) dependent fructo-kinase, whereby fructose isconverted to fructose-1-phosphate. In the liver and in the kidneys,fructose-1-phosphate is cleaved to glycerine aldehyde anddihydroxyacetone phosphate by aldolase B.

Three different types of fructose metabolism disorder are known inhumans, namely hereditary fructose intolerance, intestinal fructoseintolerance (sometimes also referred to as fructose non-absorption orfructose-malabsorption), and fructose-1,6-diphosphatase deficiency. Inaddition, there is fructosuria, which generally does not requiretreatment according to current scientific thinking.

Hereditary fructose intolerance (HFI) results from a deficiency ofaldolase B, an enzyme that is present in the intestinal mucosa, theliver, lymphocytes and the kidneys. This enzyme usually breaks downfructose-1-phosphate to fructose-1,6-biphosphate via intermediatestages. If an aldolase B deficiency is present, an excess offructose-1-phosphate occurs, leading to an inhibition of glycogenbreakdown and of gluconeogenesis and, in turn, to severe hypoglycemiawith outbreaks of sweating, tremor, vomiting and cramps after the intakeof fructose. Acidosis, kidney damage and aminoaciduria can occur if thisremains undetected. In infants, the risk ranges from hemorrhages tosudden infant death syndrome.

In contrast, intestinal fructose intolerance exhibits other symptoms. Itis wide-spread and occurs with increasing frequency, in particular inthe western industrialized nations. Intestinal fructose intolerance iscaused by a disorder of the resorption of fructose as a result of theimpairment of transport processes in the mucosa of the small intestine.The sufferer shows unclear abdominal distress/symptoms and as a resultof the bacterial degradation of the carbohydrates which have beentransferred into the colon increased intestinal production of gas. Theaffliction comprises e.g. bloated feeling, flatulences, colic-likebellyaches, aqueous diarrhoeas and noises in the bowels. Often, a wrongdiagnosis of irritable colon is made.

Fructose-1,6-diphosphatase deficiency involves a deficiency of this keyenzyme in gluconeogenesis. This causes an increase in lactate levels inthe blood after fructose exposure and fasting hypoglycemia, withlactacidosis, seizures, muscular hypotension, and coma. The developmentof fatty liver also leads to hepatomegaly.

Not all disorders of fructose metabolism necessarily lead to severefructose in-tolerance. However, even in mild disorders of fructosemetabolism, health impairments are often to be observed, which hithertocould only be influenced by a change of diet. Excessive consumption offructose containing foodstuffs can also lead to health impairments.

The above-mentioned symptoms and complaints could only be avoided up tonow by maintaining a fructose-, sucrose- and sorbitol-free diet.However, it is very difficult for those affected to keep to such a diet,since fructose is contained in all fruits and many vegetables, and iswidely used as a sweetener by the foodstuffs industry. All foods thatcontain, e.g. sucrose (household sugar) also have to be avoided. Such adiet, which is indeed very strict in the case of hereditary fructoseintolerance, is not only difficult to keep to, it is also extremelyunfavorable from a nutritional physiological point of view, andconsiderably impairs the quality of life of those affected. Not onlythose affected, but also the specialist community, consisting ofdoctors, specialists, nutritional scientists, nutritional advisers,specialist journalists, etc., have assumed for decades that there is noalternative to maintaining the diet described above. Research focused onan alternative to this diet has remained unsuccessful to date. An agentthat would make it possible to do without maintaining such a diet andwould allow the intake of fructose containing food would thus satisfy anurgent need for the many people affected that has existed for decades.It would overcome a prejudice that has been established in thespecialist world and among those affected and mean a very considerableimprovement and a dramatic step forward in the therapeutic andnutritional options in fructose intolerance, since, apart frommaintaining a diet, there has simply been no therapy available up tonow. Such an agent would also put an end to the as yet unsuccessfulefforts of the specialist world to enable those affected to eat normallyand to consume fructose containing meals without suffering side effects.The importance of such an agent becomes very clear if one also takesinto consideration that those affected by hereditary fructoseintolerance are threatened by the severest and most dangerousconsequences for their health if they, e.g. unknowingly, inadvertentlyor unintentionally consume fructose. All of this would apply all themore to an agent that additionally had no negative effects on health.

There is thus provided, in accordance with an embodiment of theinvention, a method of treating or reducing the effects in a subject ofa condition selected from fructose intolerance and impaired fructosemetabolism, the method comprising administering to a subject in need ofsuch treatment or reduction an efficacious amount of a glucoseisomerase, other than in combination with 5-D-fructose dehydrogenase. Insome embodiments, the condition is fructose intolerance that is selectedfrom the group consisting of (a) hereditary fructose intolerance, (b)intestinal fructose intolerance, (c) fructose intolerance that is due toa lack of fructose 1,6-diphosphatase and (d) combinations thereof. Insome embodiments, the glucose isomerase is administered prior to saidsubject's eating, concurrently with the subject's eating or after thesubject's eating. In some embodiments, the glucose isomerase isadministered within 60 minutes of the subject's eating. In someembodiments, the glucose isomerase is administered within 30 minutes ofthe subject's eating. In some embodiments, the glucose isomerase isadministered within 15 minutes of the subject's eating. In someembodiments, the glucose isomerase is administered within 10 minutes ofthe subject's eating. In some embodiments, the glucose isomerase isadministered within 5 minutes of the subject's eating. In someembodiments, the glucose isomerase is administered with a second enzymewhich cleaves fructose from a more complex sugar. In some embodiments,the second enzyme is selected from the group consisting of invertase,maltase and combinations thereof. In some embodiments, theadministration comprises oral administration.

There is also provided, in accordance with an embodiment of theinvention, a mammalian ingestible composition of matter selected from apharmaceutical composition and a dietary supplement, said composition ofmatter comprising a glucose isomerase, a second enzyme other than5-D-fructose dehydrogenase, and a carrier or excipient that isacceptable for use in pharmaceutical compositions, dietary supplementsor foodstuffs. In some embodiments, the composition of matter is in unitdosage form. In some embodiments, the composition of matter is in anorally administrable form. In some embodiments, the composition ofmatter is in a form selected from (a) the group consisting of a tablet,capsule, gel cap, pellet and dragee, (b) the group consisting of asolution, a suspension and a gel, optionally which is a contained in asingle-dose container and (c) powder form which is contained in asingle-dose container. In some embodiments, the composition of matter iscontained in a device operable to deliver a single dose or single dosesof the composition, such as a syringe for use with children adapted todeliver a single dose orally. In some embodiments, the glucose isomeraseis protected by a coating which is stable at pH below 4. In someembodiments, the glucose isomerase is protected by a coating whichdissolves at a pH of 5.5 or higher. In some embodiments, the compositionof matter is in unit dosage form and the coating protects the entiredosage unit. In some embodiments, the glucose isomerase ismicroencapsulated. In some embodiments, the glucose isomeraseconstitutes between 5 and 99.9% by weight of the composition of matter.In some embodiments, the unit dosage form contains between 0.01 and100,000 units of glucose isomerase activity. In some embodiments, thecomposition of matter comprises a coating which is stable at a pH below4. In some embodiments, the composition of matter comprises a coatingwhich dissolves at a pH of 5.5 or higher. In some embodiments, theglucose isomerase is not contained in an inorganic-based sol-gelbiocompatible matrix. In the context of this patent application, theterm “inorganic-based sol-gel biocompatible matrix” includes asilica-based sol-gel biocompatible matrix. In some embodiments, thesecond enzyme is selected from the group consisting of invertase,maltase and combinations thereof.

There is also provided, in accordance with embodiments of the invention,a foodstuff which comprises glucose isomerase, at least some of theglucose isomerase being present in the foodstuff in a form in which theglucose isomerase will be available in active form after ingestion ofthe foodstuff; a second enzyme other than 5-D-fructose dehydrogenase, atleast some of said second enzyme being present in said foodstuff in aform in which said second enzyme will be available in active form afteringestion of said foodstuff; and wherein, to the extent the foodstuffcontains 5-fructose dehydrogenase, the 5-fructose dehydrogenase will notbe available to act on fructose which is present in or released from thefood pulp in the digestive tract after ingestion of the foodstuff. Inthis manner, the glucose isomerase and, when present, the second enzymewill be available to act on their respective substrates which arepresent in or released from the food pulp present in the digestivetract. “Food pulp” in the context of this patent application constitutesany food, including that ingested before, concomitantly with or afterthe ingestion of the glucose isomerase-containing foodstuff, as well asthe glucose isomerase-containing foodstuff itself, that the activeglucose isomerase and, when present, active second enzyme encounter inthe digestive tract. In some embodiments, the foodstuff is selected fromthe group consisting of a cooked foodstuff, and a liquid foodstuff. Insome embodiments, the glucose isomerase is not contained in aninorganic-based sol-gel biocompatible matrix. In some embodiments, theglucose isomerase is microencapsulated. In some embodiments, thefoodstuff is substantially free of substances which are not approved fororal human ingestion. In some embodiments, the foodstuff is not a doughor a dough premix. In some embodiments, the foodstuff is not a bakedfoodstuff. In some embodiments, the foodstuff is not a bread.

There is also provided, in accordance with an embodiment of theinvention, a process for preparing a glucose isomerase-containingfoodstuff, the process comprising adding to a foodstuff glucoseisomerase in a form in which at least some of the glucose isomerase willbe available in active form after the foodstuff is ingested by asubject, whereby to obtain the glucose isomerase-containing foodstuff.In this manner, the glucose isomerase will be available to act onfructose which is present in or released from the food pulp present inthe digestive tract.

There is also provided, in accordance with an embodiment of theinvention, a kit comprising glucose isomerase and instructionsexplaining how to use the glucose isomerase to diagnose, treat, orreduce the effects of a condition selected from fructose intolerance andimpaired fructose metabolism. In some embodiments the condition isfructose intolerance that is selected from the group consisting of (a)hereditary fructose intolerance, (b) intestinal fructose intolerance(sometimes also referred to as fructose non-absorption orfructose-malabsorbtion), (c) fructose intolerance that is due to a lackof fructose 1,6-diphosphatase and (d) combinations thereof. In someembodiments the instructions instruct to administer the glucoseisomerase prior to the subject's eating, concurrently with the subject'seating or after the subject's eating. In some embodiments, the glucoseisomerase is administered within 60 minutes of the subject's eating. Insome embodiments, the glucose isomerase is administered within 30minutes of the subject's eating. In some embodiments, the glucoseisomerase is administered within 15 minutes of the subject's eating. Insome embodiments, the glucose isomerase is administered within 10minutes of the subject's eating. In some embodiments, the glucoseisomerase is administered within 5 minutes of the subject's eating. Insome embodiments the instructions instruct to administer the glucoseisomerase with a second enzyme which cleaves fructose from a morecomplex sugar. In some embodiments the second enzyme is selected fromthe group consisting of invertase, maltase and combinations thereof. Insome embodiments the instructions instruct oral administration. In someembodiments the instructions instruct to administer the glucoseisomerase other than in combination with a 5-D-fructose dehydrogenase.

There is also provided, in accordance with an embodiment of theinvention, a foodstuff which comprises glucose isomerase, wherein saidfoodstuff is not a dough or a dough premix. In some embodiments, thefoodstuff is in unit dosage form. In some embodiments, the foodstuff isin a form selected from (a) the group consisting of a tablet, capsule,gel cap, pellet and dragee, (b) the group consisting of a solution, asuspension and a gel, optionally which is contained in a single-dosecontainer, or (c) powder which is contained in a single-dose container.In some embodiments, the composition of matter is contained in a deviceoperable to deliver a single dose or single doses of the composition,such as a syringe for use with children adapted to deliver a single doseorally. In some embodiments, the foodstuff is not a baked foodstuff. Insome embodiments, the foodstuff is not a bread. In some embodiments, atleast some of the glucose isomerase is present in the foodstuff in aform in which the glucose isomerase will be available in active formafter ingestion of the foodstuff. In this manner, the glucose isomerasewill be available to act on fructose which is present in or releasedfrom the food pulp present in the digestive tract. In some embodiments,to the extent the foodstuff contains 5-fructose dehydrogenase, the5-fructose dehydrogenase will not be available to act on fructose whichis present in or released from the food pulp present in the digestivetract after ingestion of the foodstuff. In some embodiments, thefoodstuff is selected from the group consisting of a cooked foodstuff,and a liquid foodstuff. In some embodiments, the glucose isomerase isnot contained in an inorganic-based sol-gel biocompatible matrix. Insome embodiments, the glucose isomerase is microencapsulated. In someembodiments, the foodstuff is substantially free of substances which arenot approved for oral human ingestion. In some embodiments, thefoodstuff contains a second enzyme other than 5-D-fructosedehydrogenase, at least some of said second enzyme being present in saidfoodstuff in a form in which said second enzyme will be available inactive form after ingestion of said foodstuff. In some embodiments, thefoodstuff is labeled as being advantageous for persons who suffer fromfructose intolerance and/or a fructose metabolism disorder.

In some embodiments, the glucose isomerase is isolated from a naturalsource. In some embodiments, the glucose isomerase is isolated from arecombinant source.

In some embodiments, the foodstuff further comprises a second enzymewhich is selected from the group consisting of invertase, maltase andcombinations thereof.

There are provided, in some embodiments of the present invention, aneffective agent that can be used not only in milder disorders offructose metabolism, but also in hereditary and intestinal fructoseintolerance and in fructose-1,6-diphosphatase deficiency, especially inorder to enable the consumption of normally fructose containingfoodstuffs even if fructose intolerance is present. Some embodiments ofthe invention make it possible for those affected by fructoseintolerance to eat foodstuffs that they were not allowed to eat up tonow, due to their fructose content. Some embodiments provide an agentthat can reduce or prevent the occurrence of fructose intolerancesymptoms after the intake of fructose.

In some embodiments, there is provided an agent that contains glucoseisomerase, other than in combination with 5-fructose dehydrogenase. Aglucose isomerase in the sense of this application is an enzyme that isable to convert fructose into glucose. This conversion can also beachieved by a xylose isomerase. Thus, in this application, a xyloseisomerase is also a glucose isomerase. A possible method for producing axylose isomerase is described in Yamanaka, Biochimica et BiophysikaActa, issue 151 (3), 1968, 670-680, “Purification, Crystallization andProperties of the D-Xylose-Isomerase from Lactobacillus brevis” and inYamanaka, Methods in Enzymology, issue 41, 1971, 466-471, “D-XyloseIsomerase from Lactobacillus brevis”; these documents are herebyincorporated by reference.

The agent according to embodiments of the present invention can bringabout the conversion of the fructose in food or in food pulp intoglucose. The fructose so converted is thus no longer available for thebacterial metabolism in the intestines characterized by fermentation,and the likelihood of an excess of fructose-1-phosphate occurring in theliver or elsewhere is reduced. This can also prevent an increase inlactate levels in the blood.

There is provided in accordance with some embodiments an agent thatreduces the bioavailability of fructose in the human or animal body withthe help of glucose isomerase.

There is also provided in accordance with some embodiments an agentthat, with the help of glucose isomerase, reduces the amount of fructoseavailable to the human or animal body or to intestinal bacteriacolonizing therein.

There is also provided in accordance with some embodiments an agent foruse in the case of fructose intolerance, which contains glucoseisomerase.

There is also provided in accordance with some embodiments the use ofglucose isomerase in the case of fructose intolerance.

There is also provided in accordance with some embodiments the use ofglucose isomerase for the production of a product for the use in thecase of fructose intolerance.

Glucose isomerase, which belongs to the group of isomerases, is anenzyme that has the property of converting D-fructose into D-glucose andvice versa. Here, an equilibrium of approximately 50% glucose and 50%fructose is established, depending on ambient temperature. Whereasfructose is only absorbed slowly from the small intestine, glucose is asugar that is easily digested and rapidly absorbed.

Thus, without wishing to be bound by theory, it is believed that someembodiments of the invention take advantage of the phenomenon ofingested fructose being converted into glucose in vivo by glucoseisomerase that is consumed simultaneously or at least shortly before orthereafter. The glucose isomerase then attempts to achieve theabove-mentioned equilibrium by converting fructose to glucose. However,glucose is absorbed very rapidly, so that the equilibrium cannot beachieved. Theoretically, the glucose isomerase may continue to convertfructose still available in the food pulp into glucose until no furtherfructose is left. The dose of glucose isomerase may be selected in sucha way that, even if larger amounts of fructose are consumed, thereaction can take place quickly enough that substantially no fructose isabsorbed or that the amount of fructose absorbed in the meantime is toosmall to cause the known gastrointestinal complaints in the case of mildfructose metabolism disorders and intestinal fructose intolerance andthe known systemic complaints in the case of hereditary fructoseintolerance and fructose-1,6-diphosphate deficiency.

As disclosed in co-pending application Ser. No. 12/093,822, the enzyme5-D-fructose dehydrogenase (syn. fructose 5-dehydrogenase) can bringabout the conversion of the fructose in food to 5-keto-D-fructose bydehydrogenation. Thus, the fructose is changed in such a manner that itis no longer available to the metabolism of the human or animal body. A5-D-fructose dehydrogenase in this sense is an enzyme that can bringabout the dehydrogenation of fructose to 5-keto-D-fructose. Particularlyfavorable effects can therefore be achieved if glucose isomerase and5-D-fructose dehydrogenase are used in combination. A possible methodfor the production of a 5-D-fructose dehydrogenase is, for example,described in Ameyama et al., Journal of Bacteriology 1981, 814-823,“D-Fructose Dehydrogenase of Gluconobacter industrius: Purification,Characterization and Application of Enzymatic Microdetermination ofD-Fructose”, the content of which is incorporated herein by reference.

Thus, disclosed in co-pending application Ser. No. 12/093,822 is anagent that, beside glucose isomerase, additionally contains 5-D-fructosedehydrogenase. Such a combination agent can also be used in the form oftwo separate dose units, e.g. two separate tablets, one of whichcontains glucose isomerase and the other 5-D-fructose dehydrogenase.

In accordance with embodiments of the present invention, a glucoseisomerase can be used to reduce the fructose content in a foodstuff. Asdisclosed in co-pending application Ser. No. 12/093,822, a glucoseisomerase can be used in combination with 5-D-fructose dehydrogenase toreduce the fructose content of a foodstuff.

Foodstuffs in the sense of the present application also comprise, amongother things, foodstuffs for particular nutritional uses, foods forspecial medical purposes, medical foods, food supplements, dietarysupplements, dietetic food supplements, health foods, nutraceuticals,and food additives.

In some embodiments, the foodstuff may be labeled as being advantageousfor persons who suffer from fructose intolerance and/or a fructosemetabolism disorder. Such labeling need not necessarily use the exactwords “advantageous for persons who suffer from fructose intoleranceand/or a fructose metabolism disorder”; the labeling may have words tosimilar effect. For example, the labeling may state that the foodstuffis suitable for sufferers of fructose intolerance (and may name the typeof fructose intolerance); or that the foodstuff is low in fructosecontent or has a reduced fructose content, or is low in effectivefructose content, i.e., the amount of fructose present as free fructoseand fructose which can be cleaved from other molecules; or that thefoodstuff contains glucose isomerase (and may indicate that the glucoseisomerase is in active form); or that the foodstuff contains enzyme(s)that reduce the fructose content (and may indicate that the enzyme(s)lower the fructose content after consumption of the foodstuff); or thelabeling may be a symbol of approval from an organization for fructoseintolerance sufferers; or the labeling may provide instructions for useof the foodstuff as part of a regimen for treating fructose intoleranceor mitigating the effects or symptoms of fructose intolerance; and thelike. While it is contemplated that, in general, it is the packaging inwhich the foodstuff is packaged that will bear the labeling, it will beappreciated that this need not be the case, and that, for example, thefoodstuff itself may bear such labeling, or that the foodstuff may bedisplayed in a place where such labeling is also displayed, even if noton the packaging.

Embodiments of the present invention facilitate the transformation offructose in a foodstuff into a form that avoids the problems thataccompany fructose intolerance. Thus, embodiments of the presentinvention also make it possible for people affected by fructoseintolerance to consume such foodstuffs, which had to be avoided up tonow because of their fructose content.

In accordance with embodiments of the present invention, glucoseisomerase is further mentioned for use in medicine, for example as apharmaceutical composition. As disclosed in co-pending application Ser.No. 12/093,822, a glucose isomerase can be used in combination with5-D-fructose dehydrogenase in medicine, for example as a pharmaceuticalcomposition. Accordingly, in accordance with embodiments of theinvention there is provided a product which contains or consists ofglucose isomerase along with one or more other active ingredients, foruse in a medical method, in particular in a method for the therapeutictreatment of the human or animal body. In this patent application, apharmaceutical composition is a product, in particular a substance or asubstance mixture, for use in a method for surgical or therapeutictreatment of the human or animal body or in diagnostic methods that areperformed on the human or animal body. Thus, in this application,pharmaceutical compositions are also products, in particular substancesor substance mixtures, that are intended or suitable for curing,alleviating, preventing or determining fructose intolerance.

In accordance with embodiments of the present invention, a foodstuff isprovided which contains glucose isomerase in a form in which at leastsome of the glucose isomerase will exert its activity after ingestion ofthe foodstuff. In accordance with some embodiments, a foodstuff isprovided that contains glucose isomerase in an amount which issufficient to convert fructose into glucose after ingestion of thefoodstuff. In some embodiments, such a foodstuff may be produced using amethod for treating a foodstuff in which the foodstuff is placed incontact with a glucose isomerase under such conditions under which theglucose isomerase can convert fructose to glucose, provide that at leastsome of the glucose isomerase is incorporated into the foodstuff in amatter that it will exert activity after ingestion of the foodstuff. Incontrast to otherwise untreated foodstuffs, such a foodstuff willeffectively have a reduced fructose content after ingestion, and mayhave a reduced fructose content before ingestion as well, and thereforeis suitable to be consumed by persons suffering from fructoseintolerance. Thus in some embodiments, a foodstuff can be prepared by amethod in which a glucose isomerase is added to the foodstuff in amanner in which the action of at least some of the glucose isomeraseonly starts after the intake of the foodstuff. Such a foodstuff thatcontains glucose isomerase has the same taste as an untreated foodstuffand is suitable to be consumed by persons suffering from fructoseintolerance, due to the reduced fructose content which is establishedafter eating.

In some embodiments, a medical device is provided that contains glucoseisomerase. Thus in accordance with some embodiments there is provided amedical device which consists of a glucose isomerase or contains aglucose isomerase along with one or more other active ingredients. Inthis application a “medical device” means any instrument, apparatus,appliance, material or other article, whether used alone or incombination, including the software necessary for its proper applicationintended by the manufacturer to be used for human beings for the purposeof:

diagnosis, prevention, monitoring, treatment or alleviation of disease,

diagnosis, monitoring, treatment, alleviation of or compensation for aninjury or handicap,

investigation, replacement or modification of the anatomy or of aphysiological process,

control of conception,

and which does not achieve its principal intended action in or on thehuman body by pharmacological, immunological or metabolic means, butwhich may be assisted in its function by such means;

An instrument, apparatus, appliance, material or other article that doesnot achieve its intended action in or on the human body is not a medicaldevice in the sense of this application.

In the following, embodiments of the invention will be described furtherin various aspects. If the term agent is used below, this also standsfor a foodstuff, a medical device or a pharmaceutical composition.

Glucose isomerase is a compound that has been known for more than 40years and has only been used for starch saccharification to date. In theindustry, it is used for the conversion of glucose into fructose as wellas for the conversion of fructose into glucose.

The agents in accordance with embodiments of the present invention canbe taken orally before meals, with meals or immediately after meals, sothat they can exert their converting effect on fructose in the foodpulp. Preferably, the agents are taken just before meals, during mealsor immediately after meals. The agents may contain the enzyme withoutfurther additives. However, in some embodiments the agents furthercontain additives that are pharmaceutically acceptable and/or acceptablefor foodstuffs, such as extenders, binders, stabilizers, preservatives,flavorings, etc. Such additives are commonly used and well known for theproduction of pharmaceutical compositions, medical devices, foodstuffs,foodstuffs for particular nutritional uses, foods for special medicalpurposes, medical foods, food supplements, dietary supplements, dieteticfood supplements, health foods, nutraceuticals, and food additives andspecialists in this field know which additives in which amounts aresuitable for particular presentation forms. In some embodiments, theagents contain as additives dicalcium phosphate, lactose, modifiedstarch, microcrystalline cellulose, maltodextrin and/or fibersol.

In accordance with some embodiments, the agents can be added to afoodstuff before eating. They can even be added to the foodstuff at theproduction stage, with the aim of developing their effect only afterconsuming the foodstuff. This could possibly be achieved bymicroencapsulation, for example. In this way, the useable fructosecontent of the foodstuff would be reduced in a particularly advantageousway, without negatively affecting its taste. Therefore, preparationscontaining glucose isomerase are contemplated that do not release atleast some of this enzyme until it reaches the digestive tract of ahuman or animal, or let the enzyme become active in another way,especially in the stomach or small intestine. Therefore, in accordancewith embodiments of the invention, glucose isomerase could be used forexample in the production of sweets, fruit preparations (e.g. applesauce), jam, honey, chocolate and chocolate products, bakery products(e.g. biscuits and cakes), breads, pastas, vegetable dishes, potatodishes, ice cream, cereals, dairy products (e.g. fruit yoghurt andpudding), fructose containing beverages, fructose containing sauces(e.g. tomato ketchup) and fructose containing sweeteners. For dishesthat are boiled or baked, the agents could, e.g. be mixed into orsprinkled onto them after cooling.

The agents can also be added to a foodstuff to exert their effect onfructose originating from a different foodstuff. An example of thiswould be the addition of the agents to a spread so that the reduction ofthe utilizable fructose contained in the bread upon which the spread isspread occurs after ingestion of the bread, without impairing the tasteof the bread. Another example would be mixed spices.

In some embodiments, the agents also contain other active ingredients.

The invention may be formulated in any form which is suitable for theintended route of administration. For oral administration, the agentsmay be formulated in the form of capsules (coated or non-coated),tablets (coated or non-coated), capsules containing coated or non-coatedpellets, granules, or micro- or mini-tablets, tablets pressed fromcoated or non-coated pellets, dragees, or micro- or mini-tablets, gelcaps or, in liquid form, as a solution, drops, suspension or gel. Theformulation of the agent according to the present invention as a powderis particularly suitable for an admixture to a foodstuff. The powder maybe sprinkled onto a meal or it may be mixed into a pulp or a beverage.It is particularly suitable if the agent offered as bulk powder ispacked in single dosage amounts, such as in single bags or capsules, orif it is provided in a dosing apparatus. It is especially preferable ifthe agent is formulated as a powder or as granules in capsules or as atablet that are administered orally.

For oral administration, the glucose isomerase may be mixed withacceptable excipients and/or carriers. The term “acceptable carrier”relates to either a carrier for pharmaceutical use or for use in afoodstuff such as a dietary supplement which directs the activeingredient to its target site and which does not have negative effectsfor the recipient, human or animal. However, the exact form of thecarrier is not decisive.

The total amount of the carrier and/or excipients of an agent containingglucose isomerase is preferably between 5 and 99.9% by weight, morepreferably between 10 and 95% by weight and even more preferably between25 and 90% by weight of the composition.

Suitable excipients and/or carriers include maltodextrin, calciumcarbonate, dicalcium phosphate, tricalcium phosphate, microcrystallinecellulose, dextrose, rice flour, magnesium stearate, stearic acid,croscarmellose sodium, sodium starch glycolate, crospovidone, sucrose,vegetable gums, lactose, methylcellulose, povidone, carboxymethylcellulose, corn starch, modified starch, fibersol, gelatine,hydroxypropylmethyl cellulose and the like (including mixtures thereof).Preferable carriers include calcium carbonate, magnesium stearate,maltodextrin, dicalcium phosphate, modified starch, microcrystallinecellulose, fibersol, gelatine, hydroxypropylmethyl cellulose andmixtures thereof.

The various ingredients and the excipient and/or carrier are mixed andformed into the desired form using common methods. The presentation formwhich is intended for oral administration, such as a tablet or capsule,may be coated with a coating that is resistant to low pH values. Thismakes it possible for the enzyme or enzymes to be released only whenthey reach the small intestine. Also a coating may be used which is notresistant against low pH values but which provides delayed release ofthe respective enzyme at low pH values. It is also possible to preparethe agent in accordance with embodiments of the present invention ascoated (see above) pellets, granules, or micro- or mini-tablets whichcan be filled into non-coated capsules or which can be pressed intonon-coated tablets. Suitable coatings are, for example, celluloseacetate phthalate, cellulose derivatives, shellac, polyvinylpyrrolidonederivatives, acrylic acid, polyacrylic acid derivatives and polymethylmethacrylate (PMMA), such as Eudragit® (from Röhm GmbH, Darmstadt), e.g.Eudragit® FS30D (releases the active constituent or constituentsstarting at a pH of around 6.8) and/or Eudragit® L30D-55 (releases theactive constituent or constituents starting at a pH of around 5.5). Ifit is desired to release the enzyme(s) already at a lower pH value, thismay be achieved e.g. by the addition of sodium hydroxide solution to thecoating agent Eudragit® L30D-55, because in this case carboxyl groups ofthe methacrylate would be neutralised. Therefore, this coating will bedissolved, for example, already at a pH value of 4.0 provided that 5% ofthe carboxyl groups are neutralised. The addition of about 100 g of 4%sodium hydroxide solution to 1 kg of Eudragit® L30D-55 would result in aneutralisation of about 6% of the carboxyl groups. Further details aboutformulation methods and administration methods can be found in the 21stedition of “Remington: The Science & Practice of Pharmacy”, published2005 by Lippincott, Williams & Wilkins, Baltimore, USA, in theEncyclopedia of Pharmaceutical Technology (Editor James Swarbrick) andin Prof. Bauer “Lehrbuch der Pharmazeutischen Technologie”, 18thedition, published 2006 by Wissenschaftliche Verlagsgesellschaft (ISBN3804-72222-9). The contents of these documents are incorporated hereinby reference.

Other suitable pharmaceutically or dietarily acceptable carriers orexcipients include water, mineral oil, ethylene glycol, propyleneglycol, lanolin, glyceryl stearate, sorbitan stearate, isopropylmyristate, isopropyl palmitate, acetone, glycerine, phosphatidylcholine,sodium cholate and ethanol, but are not limited thereto.

The compositions for use in accordance with embodiments of the presentinvention may also comprise at least one coemulsifying compound, such asoxyethylenated sorbitan monostearate, fatty alcohols such as stearylalcohol or cetyl alcohol, or esters of fatty acids and polyols, such asglyceryl stearate, but not limited thereto.

Preferably, the agents to be used in accordance with embodiments of thepresent invention are provided in a stabilized form. Generally,stabilization methods and procedures which may be used include any andall methods for the stabilization of chemical or biological materialwhich are known in the art, comprising e.g. the addition of chemicalagents, methods which are based on temperature modulation; methods whichare based on irradiation or combinations thereof. Chemical agents thatmay be used according to the present invention include, among others,preservatives, acids, bases, salts, antioxidants, viscosity enhancers,emulsifying agents, gelatinizers, and mixtures thereof.

Conventionally, the industrial production of enzymes is performed in atechnical fermentation way using suitable microorganisms (bacteria,moulds, yeasts). The strains are recovered from natural ecosystemsaccording to a special screening protocol, isolated as pure cultures aswell as improved in their properties with respect to the enzyme spectrumand biosynthesis performance (volume/time yield). The enzymes can alsobe produced using methods to be developed in the future.

Glucose isomerase is commercially available (e.g. Novozymes A/S, Denmarkand Danisco, Denmark) and is usually prepared in a microbiological waywith the help of the microorganism Streptomyces murinus. 5-D-fructosedehydrogenase is also commercially available (e.g. Sigma-Aldrich andToyobo, Japan) and is conventionally prepared in a microbiological waywith the help of the microorganism Gluconobacter industrius. However,the invention is not limited to utilizing enzymes that are commerciallyavailable at the moment, but generally relates to enzymes that can bringabout the conversion of fructose—specifically or non-specifically—toglucose or 5-keto-D-fructose. A person skilled in the art can preparesuitable further enzymes by conventional methods, for example bymutagenesis of the gene encoding glucose isomerase which is present inStreptomyces murinus or by mutagenesis of the gene encoding 5-D-fructosedehydrogenase which is present in Gluconobacter industrius. The enzymesmay also be prepared with the help of other microorganisms, such asfungi, in sufficient amounts and the required purities, also by the useof genetic engineering methods which are common today. If it is desirede.g. to produce the enzymes with other microorganisms, the geneticinformation of a microorganism which has been found initially byextensive screening and which has also been proven as a suitable sourceof the enzyme with the desired properties can be transferred to amicroorganism which is normally used for the production of enzymes. Alsothe modification of the enzyme(s) and the production of the enzyme(s) bymeans of methods which are presently known or may be developed in thefuture in the area of industrial enzyme development and enzymeproduction, such as genetic engineering, is possible. The use and themanner of performing all these methods for developing and producing theenzyme(s) with the desired purities and activities and with the desiredproperties, in particular with respect to the stability of the enzyme(s)at various pH values, regarding the optimum of the pH value, thestability at various temperatures and temperature optimum, are wellknown to a person skilled in the art. The explanations in chapter 2(page 82 to page 130) of the textbook “Lebensmittel-Biotechnologie undErnahrung” of Heinz Ruttloff, Jürgen Proll and Andreas Leuchtenberger,published by Springer Verlag 1997 (ISBN 3-540-61135-5) describe thesemethods in detail. These methods are also described in “Advances inFungal Biotechnology for Industry, Agriculture, and Medicine” by Jan S.Tkacz, Lene Langeand try, Agriculture, and Medicine” by Jan S. Tkacz,Lene Langeand (published in 2004, ISBN 0-306-47866-8), in “Enzymes inIndustry: Production and Applications” by Wolfgang Aehle (Editor),published in 2004, ISBN 3527295925 and in “Microbial Enzymes andBiotransformations” by Jose-Luis Barredo (Humana Press 2005, ISBN1588292533). These documents are herewith incorporated into the patentapplication by reference. All this also applies to the enzymes mentionedbelow that can optionally be added to the agent according to the presentinvention.

The activity of glucose isomerase is defined in units, whereby one unitis the amount of glucose isomerase that converts 1 g of fructose toglucose in 5 minutes at a pH of 7.5 and a temperature of 37° C. from aninitial 10% solution of fructose by weight (i.e. 10 g fructose in 90 gwater).

At an enzyme activity determined according to this definition, an agentin accordance with embodiments of the present invention should containglucose isomerase in an amount or activity of 0.01 to 100,000 GIU(=glucose isomerase units), preferably 0.05 to 10,000 GIU andparticularly preferably 0.1 to 1,000 GIU per dose unit.

As described in copending application Ser. No. 12/093,822, if the agentalso contains 5-D-fructose dehydrogenase, this enzyme should be presentin an amount or activity of 10 to 5 million units, preferably 25 to 2.5million units and particularly preferably 50 to 1 million units per doseunit. One unit of this enzyme is defined as the amount that converts onemicromole of D-fructose into 5-keto-D-fructose per minute at pH 4.5 and37° C.

The wide range of the above mentioned dosages may be explained by thefact that the agent(s) may be used in connection with three verydifferent types of fructose intolerance, namely in hereditary fructoseintolerance, intestinal fructose intolerance, andfructose-1,6-diphosphatase deficiency, in their range of differentseverities, and also in milder fructose metabolism disorders.Furthermore, the different dosages also result from the fact thatstrongly varying amounts of fructose are administered to the body,depending on the respective food. The enzyme or the enzymes should beused in a sufficient quantity so that it or they develop a sufficientlyhigh enzyme activity, in other words sufficient glucose isomerase toconvert an amount of fructose consumed in a normal meal (e.g. 10-50g)—in free or bound form—into glucose.

The agent, in accordance with embodiments of the present invention, maycomprise one or more additional enzymes, such as invertase (syn.beta-fructofuranosidase or beta-fructosidase), lactase (syn.beta-galactosidase), maltase (syn. alpha-glucosidase), alpha-amylase,beta-amylase, glucoamylase, pullulanase, isoamylase, amyloglucosidase,cyclomaltodextrin glucantransferase (CGTase). These enzymes have theproperty of releasing fructose and/or glucose from fructose and/orglucose containing substances and foodstuffs—alone or in combinationwith one or more of these enzymes—, whereby the enzymes pullulanase andisoamylase also increase the efficiency of glucoamylase andbeta-amylase. All these enzymes are commercially available (e.g. BioCatInc., Troy, USA or Novozymes A/S, Denmark or Amano Enzymes Inc., Japanor Sigma-Aldrich) and, up to now, have not been used in combination withglucose isomerase in the medical/pharmaceutical field, in particular notin the case of fructose intolerance. Examples for agents in accordancewith embodiments of the present invention include Glucose isomerase incombination with invertase, or Glucose isomerase in combination withmaltase, or Glucose isomerase in combination with invertase and maltase.

For example, said invertase can release fructose from e.g. sucrose.

By the addition of one or more of these enzymes to the agent, theendogenic release of fructose from fructose-containing substances orfoodstuffs, in particular from sucrose, may also be promoted andaccelerated, so that the conversion of fructose into glucose may occurearlier. Therefore, the addition of one or more of these enzymes to theagent may have the benefit of reducing the required amount of glucoseisomerase.

The activity of invertase is measured in Sumner units (SU, assayavailable e.g. from Bio-Cat Inc., Troy, Va., USA). An SU is defined asthe amount of the enzyme which converts 1 mg of sucrose into glucose andfructose under standard test conditions within 5 minutes at 20° C. and apH value of 4.5. If the agent also contains invertase, the activity ofthe invertase per dose unit should be between 50 and 250,000 SU,preferably between 100 and 150,000 SU and particularly preferablybetween 150 and 100,000 SU per dose unit.

The activity of maltase is defined in units, wherein one unit is theamount of maltase which will convert maltose to D-glucose at a rate ofone milligram per minute at 37° C. and a pH of 4.0 in a 10% maltosesolution by weight.

Where the agent also contains maltase, the activity per dose unit shouldbe between 100 and 100,000 units, preferably between 200 and 50,000units and particularly preferably between 500 and 20,000 units.

In the case of hereditary fructose intolerance, it is particularlypreferable if, in addition to glucose isomerase, folic acid in an amountof 1 mg to 100 mg, preferably 2 mg to 50 mg and particularly preferably3 mg to 10 mg per dose unit are added to the agent(s) according to thepresent invention, as folic acid increases aldolase B activity.

It may be advantageous to add an acceptor to the agent according to thepresent invention, for example at a ratio (acceptor:substrate) of 1:1 to1:1,000, preferably at a ratio of 1:2 to 1:200 and particularlypreferably at a ratio of 1:10 to 1:50. Examples of suitable acceptorswhich may be used include NAD⁺, NADP⁺, FAD⁺, vitamins, such as vitaminC₁ vitamin E or vitamin A, ferric cyanide, ketones, aldehydes,2,6-dichlorophenol-indophenol, phenazine methosulfate, nitrobluetetrazolium (including mixtures thereof), but are not limited thereto.

It may also be advantageous to add metal ions to the agent according tothe present invention, especially cations such as Mn²⁺, Mg²⁺, Ca²⁺,Zn²⁺, Fe²⁺, Co²⁺ or Cu²⁺, including mixtures thereof, preferably at amolar ratio of 10⁻⁶ to 10⁻². For the (xylose) glucose isomerase fromYamanaka described above, a particularly suitable cation is Mn²⁺.

If the agent is added to a foodstuff before eating or during production,the activity of glucose isomerase should be between 0.01 and 20,000units, preferably between 0.05 and 10,000 units and particularlypreferably between 0.1 and 1,000 units per gram fructose in thefoodstuff.

If the agent also contains 5-D-fructose dehydrogenase and is added to afoodstuff before eating or during production, the activity of5-D-fructose dehydrogenase should be between 10 and 100,000 units,preferably between 25 and 60,000 units and particularly preferablybetween 50 and 25,000 units per gram fructose in the foodstuff.

The capsule sizes mentioned below refer to the sizes defined by CapsugelBelgium BVBA, Bornem, Belgium. The size of capsules should be chosen inaccordance with the specified formulation of the agent.

An agent in capsule form (e.g. of size 3) could contain, e.g., 15 mgglucose isomerase (activity of glucose isomerase 1 GIU/mg) and 135 mgdicalcium phosphate per capsule.

If capsules, e.g. of size 1, are used, they may contain 50 mg glucoseisomerase (activity of glucose isomerase 1 GIU/mg), 5 mg folic acid and150 mg maltodextrin per capsule.

A further example of a composition for the production of capsulesconsists of capsules of size 3 that contain 15 mg glucose isomerase(activity of glucose isomerase 1 GIU/mg), 75 mg 5-D-fructosedehydrogenase (activity of 5-D-fructose dehydrogenase 90 units/mg) and60 mg dicalcium phosphate per capsule.

A further example of a composition for the production of capsulesconsists of capsules of size 00 that contain 300 mg glucose isomerase(activity of glucose isomerase 1 GIU/mg) and 170 mg dicalcium phosphateper capsule.

A unit dosage form in accordance with embodiments of the invention mayfor example contain between 0.01 and 100,000 GIU (=glucose isomeraseunits) and between 1 mg and 100 mg folic acid per dose unit. Inaddition, suitable additives in the required amount may be used.

An agent in accordance with embodiments of the invention can be madeavailable for medical purposes and non-medical purposes, e.g. as apharmaceutical composition, medical device, foodstuff, foodstuff forparticular nutritional uses, food for special medical purposes, medicalfood, food supplement, dietary supplement, dietetic food supplement,health food, nutraceutical, or food additive.

In accordance with embodiment of the invention, an agent can be used toconsiderably alleviate or eliminate the symptoms and health impairmentscaused by fructose intolerance. The invention presented here is suitablefor use in the case of fructose intolerance and for the therapeutictreatment of fructose intolerance.

The invention claimed is:
 1. A method of treating fructose intoleranceor reducing the effects of fructose intolerance, the method comprisingadministering to a subject in need of such treatment or reduction aneffective amount of an isolated glucose isomerase, wherein said glucoseisomerase is not administered in combination with5-D-fructose-dehydrogenase.
 2. The method of claim 1, wherein saidfructose intolerance is not hereditary fructose intolerance.
 3. Themethod of claim 1, wherein said fructose intolerance is intestinalfructose intolerance.
 4. The method of claim 3, wherein said intestinalfructose intolerance is caused by a disorder of fructose absorption. 5.The method of claim 3, wherein the treatment of fructose intolerance orreduction of effects of fructose intolerance comprises reducing orpreventing the occurrence of fructose intolerance symptoms selected froma feeling of bloating, flatulence, colic-like stomach ache, waterydiarrhea and bowel sounds.
 6. The method of claim 1, wherein thetreatment of fructose intolerance or reduction of effects of fructoseintolerance comprises (a) reducing the bioavailability of fructose inthe body of the subject or (b) reducing the amount of fructose availableto the body of the subject or to intestinal bacteria colonizing therein.7. The method of claim 1, wherein the treatment of fructose intoleranceor reduction of effects of fructose intolerance comprises reducing orpreventing the occurrence of fructose intolerance symptoms in thesubject after the intake by said subject of fructose or a substance orfoodstuff containing fructose in pure form or from which fructose can bereleased in the digestive tract.
 8. The method of claim 1, wherein saidglucose isomerase is contained in a pharmaceutical composition or afoodstuff.
 9. The method of claim 8, wherein said glucose isomerase iscontained in a pharmaceutical composition.
 10. The method of claim 8,wherein said glucose isomerase is contained in a foodstuff.
 11. Themethod of claim 10, wherein the foodstuff is selected from the groupconsisting of (a) foodstuffs for particular nutritional uses; (b) foodsfor special medical purposes; (c) medical foods; (d) food supplements;(e) dietary supplements; (f) dietetic food supplements; (g) healthfoods; (h) nutraceuticals; and (i) food additives.
 12. The method ofclaim 11 wherein the foodstuff is a dietary supplement.
 13. The methodof claim 1, wherein said glucose isomerase is administered orally. 14.The method of claim 13, wherein the glucose isomerase is administeredprior to said subject's eating, concurrently with said subject's eatingor after said subject's eating.
 15. The method of claim 13, wherein theglucose isomerase is formulated in a form selected from the groupconsisting of (a) coated or non-coated capsules (b) coated or non-coatedtablets (c) capsules containing coated or non-coated pellets, granules,or micro- or mini-tablets (d) tablets pressed from coated or non-coatedpellets, dragees, or micro- or mini-tablets (e) gel caps (f) a solution,(g) drops, (h) a suspension and (i) a gel.
 16. The method of claim 13,wherein the glucose isomerase is formulated as a powder.
 17. The methodof claim 15 wherein the coating of the capsules, tablets, pellets,granules, micro- or mini-tablets and dragees is selected from the groupconsisting of cellulose acetate phthalate, cellulose derivatives,shellac, polyvinylpyrrolidone derivatives, acrylic acid, polyacrylicacid derivatives and polymethyl methacrylate.
 18. The method of claim 1wherein said glucose isomerase is administered with at least one metalion.
 19. The method of claim 18, wherein the metal ion is selected fromthe group consisting of Mn²⁺, Mg²⁺, Ca²⁺, Zn²⁺, Fe²⁺, Co²⁺, Cu²⁺ andmixtures thereof.
 20. The method of claim 1 wherein the isolated glucoseisomerase is a xylose isomerase.
 21. The method of claim 1, wherein saidglucose isomerase is administered with a second enzyme which cleavesfructose from a sugar which is more complex than fructose.
 22. Themethod of claim 21 wherein said second enzyme is selected from the groupconsisting of invertase, maltase and combinations thereof.
 23. Themethod of claim 3, wherein the treatment of fructose intolerance orreduction of effects of fructose intolerance comprises reducing orpreventing the occurrence of an increase in the production of intestinalgases.